CA2084408C

CA2084408C - Illumination system and method for a high definition light microscope

Info

Publication number: CA2084408C
Application number: CA002084408A
Authority: CA
Inventors: Gary Greenberg
Original assignee: Edge Scientific Instrument Co LLC
Current assignee: MICRO SCIENCE TECHNOLOGIES
Priority date: 1991-04-19
Filing date: 1992-03-20
Publication date: 1997-11-11
Anticipated expiration: 2012-03-20
Also published as: US5345333A; ES2041229T1; AU650985B2; CA2084408A1; KR100270979B1; DE535218T1; WO1992018894A1; EP0535218A1; AU1915792A; EP0535218A4

Abstract

An illumination system and method for increasing resolution, sharpness, depth of field, and perception of depth for a transmitted light microscope including a condenser lens (12) having an optical axis (13), an objective lens (14) having an optical axis (16), and an eyepiece system (41) wherein a light beam path shift device (23 and 24) enables two or more separate light beams (28 and 32) to be focused onto the condenser lens (12) along paths (28a and 32a) that are not coincident with the condenser lens optical axis (13) and that produce beam exit paths (33 and 34) from the condenser lens (13) which are at maximum oblique angles relative to the optical axis (16) of the objective lens (14).

Description

WO 92/18894 PCr/l~S9~/02'60 ,, 2~8~0~
ILLUMINATION SYSTEM AND METHOD ~OR A
HIGH DEFINITION LIG~IT MICROSCOPE

BACXGROUND OF T~ INVENTION
1. F ield of the lnve~tion This invention relates to illumination systems for transmitted light microscopes utilizing condenser lens means and more particularly to such systems that utilize illuminating light directed 10 at an oblique angle relative to the optical axis of the microscope objective .
2. The Prior Art The use with microscopes of what is commonly refereed to as "oblique light" was of interest towards the end of the last century and the beginning of this, but the many devices designed for that purpose, although ingenious in some cases, have failed to survive.
See The IntelIigent Use of the Microscope, Oliver, C.W., Chemical Publishing Co., 1953.
Oliver carefully limits his meaning of "oblique light" to the "use of a narrow cone or beam of rays directed upon the object lspecimenl from any direction other than the optical axis provided that it enters the object glass." Ibid. at 94. In this way he excludes from his discussion those systems that use rays directed onto a specimen from a direction other than the optical axis but which do 25 not enter the object glass as well as systems where the light does not enter the objective lens at an angle (such as systems that merely tilt the specimen stage~. Illumination provided by systems in which the primary beam does not enter the objective is generally known and commonly refereed to as "dark field" illumin~tion as more fully discussed in Photomicrography a Comprehens~ve Treatise Loveland, R.P., John Weily & Sons, Chapter 12. Although the present invention utili~es true oblique lighting as that term is used by Oliver, and is thereby clearly distinguishable from "dark field" systems, a brief description of "bright field" and "dark field"
illumination will help to differentiate and more fully highlight the wo 92/1889~ PCr/~Sg2/02~60 attributes of the present invention. 2 ~ O S
Illumination systems that direct rays onto a specimen along the optical axis create "bright field" illumination, so named because the rays passing through the field surrounding the specimen arld 5 entering the microscope objective are unimpeded and thus bright compared to the rays attenuated by passing through the specimen.
In a "dar~ field" system, the relative brightness is reversed by directing only light rays onto the specimen field which are angled relative to the optical axis and directed to fall outside the objective 10 aperture. All of the light passing through the specimen field surrounding the specimen is unimpeded and thus does not enter and is therefore not "seen" by the objective. Some of the light directed onto the specimen will be scattered. however. into secondar~ light rays, some of which will enter the objective ( and 15 be "seen"). Thus, the object appears brighter than the surrounding dark field. Such a system is described in U.S. Pat. No. 4,896,966.
The prior art contains a number of systems that combine "bright field" and "dark field" illllmin~tion for use both together and selectively, as illustrated in U. K. Pat. No. 887,230, and U.S. Pat. No.
20 4.601,551. In all of these systems the primary ill~1min~ting light is either aligned with the optical axis or angled to fall outside of the objective aperture.
The invention of U.S. Pat. No. 3,876,283, teaches the use of a system which uses true oblique lighting. by use of a prism located 25 on the optical axis of a microscope condenser to laterally off-set an axial illumination beam to a path separate from the optical axis so as to direct the beam onto an off center location on the condenser lens. When a light beam parallel to the optical axis enters an off center location on a condenser lens, the beam will exit the lens at 30 an angle to the optical axis. The dëgree of the angle is a function of the displacement of the beam from the center of the lens. When, as in patent '283, the angle is within the objective aperture, the system produces true oblique lighting as defined by Oliver (the light is "seen" by the objective~. In order to achieve the maximum 35 oblique angle for the beam it must exit the condenser lens at or . .

wo92/18894 ~ i 4 0 8 Pcr/~is9~/02260 very near its peripherv at an angle that is just within the objective aperture. While the teachings of patent '283 make this possible (by adding a wedge shaped prism to the plano prism shown), each different condenser and objective combination will require a 5 different pair of prisms to achieve a maximum angle. Otherwise.
depending on the characteristics of the objective lens and condenser lens being used, it may be necessary ~vith the system of patent '283 to direct the laterally off-set beam onto the condenser lens at a location inwardly of its periphery in order to have the l 0 resultant exit angle within the objective aperture. In such cases the maximum possible oblique angle will not be realized and, as will be explained below. the maximum resolution power of the system will not be achieved.
In patent '283. the location of the illuminating beam (between 15 15 and 17) and beam path shifting means 23 (prism) on the optical axis limits the system by permitting the use of only a single illumination beam.
The references cited above are typical of the prior art in that they fail to recogni~e the real potential of oblique lighting to 20 enhance resolution. Patent '283, in fact. does not acknowledge the resolution enhancing potential of oblique light but instead cites as a reason for its use the casting of shadows to hi~hlight uneven areas of the specimen. It is not. therefore, necessarily an object or desi-deratum of patent '283 to provide a maximum oblique angle (for 25 example, too much shadowing might obscure details). But, one of the requirements of realizing the full potential of oblique lighting to dramatically enhance resolution is that the angle of the oblique light be maximized. For a single beam system. maximum resolution is achieved for a given condenser lens/objective lens combination 30 by having the illumination beam exit the condenser lens' periphery so that the light ill-lmin~ting the object is at a maximum oblique angle and still within the objecffve aperture. By making it possible to adjust the angle at which the beam exits the condenser lens independently of the location where it exits. the angle of the light 35 (relative to the optical axis of the objective lens means) wo 92/18894 Pcr/uss2/o2~6o 2~8~4~8 illuminating the specimen can be fuIly maximized. Likewise, by being able to adjust the location where the beam exits the condenser independently of the angle at which it exits, any condenser can be used to its fullest potential. With the ability to so 5 adjust the angle and location of the beam exiting the condenser lens, a large condenser lens (high numerical aperture) can be used to achieve maximum oblique lighting for most objective lenses.
The present invention teaches that the essential requirement for realizing the maximum potential of true oblique lighting is the 10 ability to direct two or more separate and distinct light beams onto the condenser wherein each beam is at the maximum angle to the objective axis that permits the ill1]min~tion to enter the objective.
This. of physical necessity, requires that the beam shifting means be located off the optical axis of the condenser. In addition, the 15 present invention overcomes the anisotropy that is found in prior art oblique illuminating systems.
In addition,the present invention teaches a real time, 3-D
system using multiple beams which goes far beyond what can be achieved with a single beam, such as that described in U.S. Patent 20 4,072.967. Patent '967 teaches how to achieve a 3-D image using a microscope with a single condenser lens and a single objective lens, by placing complimentary filters across the left and right halves of the condenser lens and placing a complementary filter set in the binocular eyepieces. With this type of system the degree of 25 parallax is fixed. Futhermore, there is very little disparity in parallax between the left and right images, especially at the center of the image field. In contrast, with the present invention the left and right images are independently controlled and the degree of parallax between them can be easily adjusted to match the type of 30 objective being employed and the type of specimen being viewed.
In addition, there is another and possibly even more important advantage with the present invention, which is the ability to achieve a greater depth of field without loss of resolution, as is more fully explained below. This is a critical prerequisite for producing a 3 5 sharp 3-D image.

wo 92/18894 PCr/us92/02260 ~5~
2~8~
SUMMARY 0~ THE INVENTION
The present invention resides in the ill1tmin~tion system for a transmitted light microscope characterized by condenser lens 5 means (which can be comprised of several lenses) and an objective lens means (also possibly comprised of several lenses). The object or specimen to be illuminated is located between the condenser and the objective.
The diffraction theory of microscopic vision teaches that l o when examining with transmitted light an object having very closely spaced structural details such as the markings of the diatom Amphtpleura pellucida. the image of a single point or line of detail will consist of a central beam surrounded by a number of spectra (sometimes referred to as orders of diffraction wavelets). The 15 number and arrangement of the spectra depend on the pattern of the markings, and the wave length of light being used. The distance of the diffraction wavelets from the central beam is greater the finer the markings on the specimen (the smaller the spacing bet~veen structural details).
The diffraction theory further teaches that in order to obtain any image of the specimen it is necessary to collect and recombine at least one order of wavelets with the central beam. The more successive orders of wavelets recombined with the central beam the more the resolution and sharpness of the image increases.
Using an axial illuminating beam on an object such as the diatom Amphipleu~a pellucida. creates spectra that are so far out from the central beam that the highest existing aperture is insufficient to include any of them. The specimen's markings remain unresolved and thus invisible.
-: The use of oblique lighting can result in the inclusion of one or more orders of wavelets for a specimen which~when illuminated by axial lighting casts all of the orders of wavelets beyond the objective. The greater the angle of the oblique light the greater the number of orders of wavelets included within the objective aperture 35 and thus the greater the resolving power of the system. In fact.

wo 92/18894 PCr/~iS92/02260 -6- 2~)8~4~8 both the resolution as well as the sharpness of the image can be significantly increased compared to axial illumination, because the optimal oblique illumination will place the zero order wavelet near the edge of the objective aperture and thus, the objective car 5 recombine more orders of diffraction wavelets for any given structural detail.
Accordingly, it is a principal object of the present invention to provide an illumination system and method for a transmitted light microscope which produces oblique lighting having the 10 maximum angle possible for the lenses used therebv enhancing the microscope's resolving power and sharpness of image.
In conjunction with the object stated above is an object of the invention to utili~e the entirety of the beam or beams directed onto the condenser as illumination sources for the specimen. That is to 15 say. that the present invention, unlike so much of the prior art.
does not use a mask on the condenser or between the condenser and the specimen to create an oblique light beam from a small portion of the beam initially directed onto the condenser.
It is a further object of the invention to provide for a 20 transmitted light microscope having a condenser lens, an illumination system which produces an oblique light beam which is independently selectively adjustable in both the location and angle at which it exits the condenser lens. - -While the use of a single illuminating beam according to the 25 present invention achieves results which can surpass the prior artin terms of resolution, and is within the scope of the invention, the maximum potential of oblique lighting is achieved in the present invention when a plurality of independent beams are used.
Specifically, while a single beam system produces enhanced 30 resolution, it does so predominantly along the direction of the beam axis (projected onto the specimen plane). Furthermore. at 90 degrees to that axis there is a significant decrease in resolution and sharpness. For example, in order to see the detailed pattern of Amphipleura PelLucida the specimen must be rotated on the stage 35 so that the markings are oriented along the axis of the oblique wo 92/18894 PCr/~,Ss2/02260 '~O~f 4~
illuminating beam. As the specimen is rotated away from that optimal position, the markings become less distinct and finally disappear altogether. As the specimen is rotated further, the markings become visible again as the orientation approaches 180 5 degrees. This is a result of the fact that while a single oblique beam increases resolution along an X dimension, it decreases resolution along the perpendicular Y dimension. If, however, two oblique beams illuminate a specimen so that their angle of orientation is 90 degrees apart, then the image resolution and sharpness is 10 increased in both the X and Y dimensions. Enhanced resolution over essentially the entire specimen plane is achieved using multiple oblique illuminating beams radially spaced about the optical axis of the condenser. As a result, very fine structural details such as the markings on Amphlplewa Pelluc~la can be seen 15 regardless of how the specimen is oriented on the stage.
When multiple beams are used, enhanced resolution is derived not only from the benefits of oblique illumination but also from the overall increase in the system's N. A. (numerical aperture) that results from multiple beams following different oblique paths 20 from the condenser to the objective. That is, the "working" N.A. of the condenser beam is increased beyond its normal potential because a highly oblique beam of light will exit the condenser lens at a greater angle than will a normal axial beam. The increased exit angle will only be on one side of the condenser while the exit angle 25 will be deficient on the opposite side of the condenser. If however, a second oblique light beam is directed into the condenser at the opposing angle relative to the first beam, then both sides of the condenser will project an exit beam with a greater angle than . would be possible with a single central light beam. Thus, multiple 30 oblique light beams can be directed into a condenser lens at -opposing angles relative to the optical axis such that the resulting exit beams will combine to form an overall increase in the aperture of illumination and thus, an increase in the overall resolution of the system. The final resolution of the image is dependent on the N.A.
35 of the system. For microscopes using an objective lens along with a wo 92/18894 Pcr/~sg2/o2~60 -8- 2~
condenser lens, the N.A. of the system will be the combination of the N.A. of objective and condenser lenses.
Thus, another object of the invention is to provide an illumination system and method for a transmitted light microscope 5 utilizing a plurality of independent, separate illuminating light beams directed onto a condenser wherein each light beam follows a different oblique angled exit path to the objective (relative to the objective's optical axis).
Another object of the invention is to provide an ill1lmination l o system for a transmitted light microscope utilizing a plurality of independent separate illuminating light beams directed onto a condenser wherein the exit path of each light beam from the condenser is independently adjustable in both its location and angle. Such a system enjoys, in addition to the advantages already l 5 stated. the advantage of being able to significantly increase the depth of field without degradation of resolution.
It is well known that in a conventional illumination system for a microscope, reducing the condenser aperture to increase depth of field and contrast, reduces resolution. A known alternative 20 method for increasing depth of field is to slightly under focus the condenser lens (keeping the condenser aperture fully open) while closing a field stop iris to increase depth of field. If a single illuminating beam is used, whether it be axial or oblique, then the increase in depth of field will be accompanied by a decrease in 25 resolution. In the present invention, multiple oblique beams are directed onto the condenser so that even when the field lens aperture is reduced to increase depth of held and contrast, resolution is not degraded. This follows because overall aperture of illumination at the condenser lens, which continues to receive and 30 transmit light beams from its full aperture, has not been reduced.
Put'in another way, the hnal image is the combination of multiple imàges, each with extended depth of field created by an array of pre-apertured oblique illuminating beams. which have an additive effect on the overall aperture of illumination. ~ -Yet another object of the invention is to provide means for .. . . .. ...

~ ~ ~ 8 ~
g using double oblique lighting in a transmitted light microscope having a condenser lens which produces enhanced resolution and real time 3-D viewing with extended depth of field.
By directing separate independent illuminating light beams 5 onto the condenser, it is possible in the present invention to manipulate each beam independently if desired, such as by interposing complementary filters and thereby produce true. real time 3-D viewing. The interposition of polanzing filters in the path of one or more beams perrnits a variety of effects, such as selective 10 shadow rotation, to be achieved at the same time that enhanced resolution is realized.
Other objects of the present invention will in part be obvious and will in part appear hereafter.
A significant part of the present invention teaches how to 15 reali~e the maximum potential of oblique illumination by directing two or more separate and distinct oblique light beams into the condenser lens in a variety of configurations in order to achieve results which would not be possible with a single illuminating beam.
Some of those configurations will be illustrated and their 20 advantages discussed. However, there are other possible configurations that will not be specifically discussed but still fall within the scope of these teachings.

BR~EF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, aspects and advantages of the invention will be better understood from the following detailed description of the preferred embodiment of the invention with reference to the drawings in which:
Figure lA is a schematic diagram of microscope optical 3~ elements (including a condenser lens and an objective lens) wherein the illumination path is coincident with the axes of the lenses;

fa~

Figure lB is a schematic diagram of the microscope optical elements of Figure lA wherein the illumination path is parallel to but not coincident with a condenser lens and oblique to the objective lens;

Figure lC is a schematic diagram of the microscope optical elements of Figure lA wherein the illumination path is non-coincident with and oblique to the axes of both lenses;

Figure lD is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of Figure lA;

Figure lE is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of Figure lB;

Figure lF is a wave diagram illustrating the relative number of orders of wavelets that can be seen by the objective lens by the illumination arrangement of 2~ Figure lC;

Figure 2 is an optical schematic illustration of a two beam embodiment of the invention;

Figures 2A and 2B are plan views illustrating two possible mirror arrangements for the embodiment of Fig. 2;
Figure 2C is a plan view illustrating a three mirror;

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Figure 2D is a plan view illustrating a four mirror configuration;

Figure 2E is a plan view illustrating six mirror configuration;

Figure 3 is an isometric, optical schematic illustration of a three beam embodiment of the invention;

Figures 3A, 3B and 3C are plan views of the mirrors of Fig. 3 in varying arrangements;

Figure 4 is an isometric illustration of a beam shift means of the invention for mirrors;

Figure 5 is an optical schematic illlustration of the embodiment of Figure 2 with the mirrors replaced by fiber optic guides; and Figure 6 is an isometric illustration of a beam shift means which is the same as that shown in Figure 4 but with the mirrors replaced by fiber optic guides.

An important aspect of the present invention is best described with reference to Figures lA - lC wherein a light beam path shift means (mirror) 11, a condenser lens means 12 having an optical axis 13, an objective lens means 14 having an optical axis 16, and a specimen support stage 17 disposed between the condenser 12 and the objective 14 and defining a specimen plane 20, are the basic components of a microscope illumination system. The support stage 17 holds a specimen (not shown) to be illuminated by light beam 18 from a light beam source means (not , f shown). The axis 13 of the condenser 12 and the axis 16 of the objective 14 are shown as being coincident which is the most common arrangement for transmitted light microscopes. Such axial coincidence is not required by the present invention, however, which is equally operative in a system where, for example, the condenser is tilted relative to the objective. Although both the condenser meansl2 and the objective meansl4 are each shown diagrammatically as a'single lens, it will be understood by those skilled in the art that the condenser means and the objective means may be comprised of multiple elements as well as other optical devices known in the art.
When as shown in Figure lA. the mirror 11 is positioned on the condenser axis 13 and disposed at a 45 degree angle relative to the initial path 19 of beam 18. which is normal to the condenser axis 13, the path 21a of the beam after being shifted by the mirror 11 will fall along the axis 13.
Unless otherwise stated. Iines indicated as representing a wo 92/18894 PCr/liss2l02260 2~8~
beam path such as 19 and 21a, are schematic representations of a bearn's axis. In reality, of course, a beam has an envelope which can be converging, diverging or parallel. An understanding of the present invention is best facilitated, however, by following the path 5 of a beam's axis.
As is well known, a beam incident a condenser such as 12 along its axis 13 will emerge from the lens along an aYial path 22a.
For the arrangement of Figure IA the beam path 22a will pass through the specimen plane 20 at right angles thereto and include 10 the objective lens 14 along its axis 16. Figure lA represents a typical "bright field" illumination system.
When mirror 11 is laterally displaced from the a~s 13 of condenser 12 while being maintained at a 45 degree angle, as shown in ~igure lB. the shifted beam path 21b remains parallel to 15 condenser axis 13 but is laterally displaced therefrom. The effect of the beam path 21b entering condenser 12 at an off axis location is to create an angle B between the exit beam path 22b and the objective axis 16. However, the exit location of the beam 22b from the condenser means 12 is not laterally displaced from the 2 0 condenser optical axis 13.
Since the specimen plane 20 is at right angles to the objective axis 16 the bearn path 22b will be angled or oblique to a specimen in the specimen plane 20. For the purposes of the present invention, however, the important relationship is the angle 25 ~ between the exit beam path 22b from the condenser 12 and the optical axis 16 of the objective 14. The advantages of the present invention do not, for example, accrue from a system that creates an oblique angle between the specimen plane and the illuminating beam path by tilting the specimen stage while at the same time 30 allowing the illuminating beam to travel a path that is parallel to the objective axis. Such an arrangement still produces standard "bright field" illumination enhanced only by some possible shadowing.
Referring to Figure lC, mirror 11 is inclined relative to the 35 path 19 of incident source beam 18, to be greater than 45 degrees .

wo 92/18894 PC~tUS92!02'60 -12~ 8~4Q~
-(50 degrees for example), causing an angle of reflectance for the bearn that sets the beam path 21c to the condenser means 12 at an angle Q relative to the optical axis 13 of the condenser means. The effect of beam path 21c entering condenser 12 at an angle Q is to 5 laterally shift the location of the exit beam path 22c from the center of the condenser lens means 12 to some location nearer the periphery. Thus, by changing the angle of the mirror 11 relative to the beam path 19, as well as laterally displacing it off of axis 13, as shown in Figure lC, the beam path shift means 11 is operative not 10 only to control the angle of the exit path 22c but its location on the condenser 12 as well. Under these circumstances, the angle of oblique illumination is increased (angle '~1 in Fig. lC is greater than angle ~ in Fig. lB). Thus, the angle of the exit beam path 22c from the condenser 12 is a function of the lateral (radial from axis 13) 15 displacement of the incident beam path 21c, while the lateral (radial from axis 13) position of the exit beam path 22c on the condenser 12 is a function of the angle of the incident bearn path 21c relative to the axis 13 of the condenser means 12.
One of the im.portant features of the present invention is the mani-20 pulsyion of the beam. path shift means 11 to m~;m;Y.e the angle of the exit beam from the condenser within the limits of the aperture of the objective. This will depend on the specification of the lenses, such as the focal length, working distance and numerical aperture.
The nU~;mm oblique angle of the beam path 22c 25 relative to the objective axis 16 within the objective aperture, is achieved by having the beam path exit the condenser at or very near the edge of the condenser lens 12. This is achieved by varying the angle of mirror 11 relative to the source beam path 19 and thereby the angle Q of the beam path 21c to the 30 condenser. At the same time, in order to create true oblique lighting, all or a portion of the beam must enter the objective, requiring that for a particular objective, the exit beam path from the condenser be at a particular angle as well as location. And, as explained above, the angle ~1 is varied as a function of the radial 3S location of the mirror 11 and thereby the radial location of the wo 92/18894 Pcr/~s92/0~260 entering beam path 21c relative to the condenser optical axis 13.
One of the advantages that accrues to the present invention is that condensers of maximum size can be advantageously used in most systems since the beam path shift means permits the angle Q
5 of the exit beam path from the edge of the condenser. to be shifted until it includes the objective. In this way, the best glass can be used and the maximum beam path angle achieved with the result of greatly enhanced resolution. Furthermore, in the present invention, unlike prior art systems, most of the optimally angled 10 light beam can enter the objective rather than merely just an edge or small portion of the light cone, thereby creating the brightest possible image for the available light.
W~ile the single beam system described above is capable of greatly enhancing a microscope's resolution, the improved 15 resolution is primarily along the direction of the axis of the illuminating beam (as projected onto the specimen plane), with the resolution along a direction 90 degrees thereto being significantly degraded.
Resolution and sharpness are ultimately dependent upon the 20 number of orders of diffraction wavelets that can be collected and recombined by the objective lens. Figs. lD,lE and lF illustrate the relative number of orders of wavelets that can be seen by the objective lens under the illuminating conditions shown in Figs. lA, lB and lC, respectively. In Fig. lE, which corresponds to the 25 oblique illuminating conditions of Fig. lB, the objective lens collects and recombines more orders of diffraction wavelets 25 than shown in Fig. lD which corresponds to the axial illuminating system of Fig. lA. However, the increase in the order of wavelets collected in the X dimension is linked to a decrease in the order of 30 wavelets collected in the Y dimension. This increase (or decrease) in resolution relative to the resolution attainable with axial illumination, is proportional to 2 times the cosine of angle 0, where angle 0 is the angle of orientation of the specimen (not shown) relative to the axis of the oblique illumination. Angle 0 35 ranges from O to 90 degrees, where O degrees is the X dimension ~ '0 92/18894 PC r/us92/o226o (or the axis of oblique illumination) and 90 degrees is the Y
dimension.
In Fig. lF, which corresponds to the maximum oblique illuminating conditions, as shown in Fig. lC, the number of 5 diffraction wavelets 25 collected and recombined by the objective is even greater than the number attainable with the oblique illuminating conditions shown by Fig. lB and lE. This results from the fact that the objective lens is viewing the wave front at such a highly oblique angle that the spacing of the wavelets appears 10 foreshortened and so more wavelets can be seen by the objective.
This additional increase in resolution is proportional to the sine of the angle between the axis of the oblique illuminating beam and the optical axis 16 of the objective lens means.
Thus, there is an increase in resolution that is related to the 15 amount of lateral displacement of the illuminating beam and there is also an increase in resolution that is related to the angle of the illuminating beam relative to the optical axis. The total increase in resolution is the combined effect of both of these elements.
One of the outstanding features of the present invention is 20 that the illumination beam shift means (i.e. mirror 11) is located off the condenser axis thereby permitting a plurality of such beam shift means to operate within the system simultaneously. Thus, improved resolution over the entire specimen plane can be achieved by utili~ing a plurality of illuminating beams positioned to have their 25 respective axes at selected angles to one another.
Referring to Figure 2, a pair of beam path shift means in the forrn of mirrors 23 and 24 disposed off the optical axis 13 of condenser 12 permit the system to operate with two independent illuminating beams to the condenser lens means 12. A light beam 30 source means (lamp) 26 directs a light beam 27 along a source beam path 28 that includes the beam path shift means 23.
Similarly, a light beam source means (lamp) 29 directs a light beam 31 along a source beam path 32 which includes beam path shift means 24. Mirror 23 shifts the direction of beam path 28 to path 35 28a which leads to condenser 12- Mirror 23 is disposed a wo 92/18~94 PCT/~S92/0~'60,_ - 1 5~
distance radially away from the condenser axis 13 and at an angle rt relative to its incident light beam 27 which produces the exit beam path 33 from condenser 12 to emerge from the edge of the lens at the m~mum angle which leads to the objective 14.
Similarly, mirror 24 shifts the direction of beam path 32 to path 32a which passes through condenser 12. Mirror 24 operates in precisely the same way as mirror 23 to produce the desired exit beam path 34 from condenser 12.

The relationship of the locations of mirrors 23 and 24 lO relative to axis 13 is shown in Figure 2A, but can be different depending on the results desired. For example, the shift means can be disposed in essentially opposing relationship (180 degrees apart) for 3-D viewing purposes as shown in Figure 2A, or at essentially nght angles (9O degrees apart) as shown in ~igure 2B, to 15 achieve the best overall resolution for a two beam system.
Resolution over the entire specimen plane is improved by increasing the number of beams. A three beam system as shown in F'igure 2C, where the beam shift means 30 are evenly angularly spaced (120 degrees apart) about axis 13, provides improved resolution over the entire specimen plane. Increasing the number of beams even further to ~our (Figure 2D) or as many as six (Figure 2E) will prod~ce even better results. Because of the off axis placement of the beam shift means, numerous other arrangements of mirrors and spacing are possible to meet specific needs.
For purposes of the present invention, the source of light beams 27 and 28 (Fig. 2) can be from separate independent light beam sources as shown, or from a light beam source means providing a single light beam which is split by beam splitting means (not shown) which are well known in the art. More important than the 30 source of the light, are the multiple beams 27 and 31 directed along separate, independent paths to the condenser, and the resultant exit beam paths 33 and 34 which do not fall along the optical axis 16 of the objective lens means 14.
Likewise, while mirrors provide one means of beam shifting, 35 other means exist, such as prisms, and the fact that all such means 2 ~ ~ ~ 4 ~ ~

are not shown does not mean that any of them are excluded from the invention. The present invention, in ~act, encompasses an arrangement of separa~e micro light sources 101, as could be provided using fiber optics 102 (see Figure 5), with the beam shift means comprising mechanical or electro- mechanical means 103 (see Figure 6) for positioning and directing these light sources. In all cases, the invention is manifest by separate, independent, light beams directed to the condenser means.
] o Additionally, for the purposes of the present invention, the beam shift means is shown to be adjustable in order to accommodate a large variety of different objective lenses. However.
with a given objective lens/condenser lens combination. there is no necessity for an adjustable beam shift means, and a fixed or pre-adjusted beam shift system would suffice. Thus, the present invention includes such fixed systems known in the art that will direct light beams into a condenser lens at the appropriate location and angle of orientation.
One of the outstanding features of the multiple beam embodiment of the present invention is the intensity of light available to illuminate the specimen at the specimen plane 20.
Unlike prior art devices that create angled light beams, the present invention does not require the use of masks or other light occluding devices. Thus, the present invention makes it possible to 2 5 utili~e virtually all of the light from the light beam source means for illumination of the specimen. While the light beam source means has been shown schematically as a light bulb, it will be understood by those skilled in the art that the light beam source means may include any suitable source of light as well as lens means and other 30 optical devices well known for the purpose of fumishing object illuminating radiation.
Another important feature of the multiple beam embodiment is that it is able to overcome the anisotropy that is inherent in all oblique illuminating systems known in the prior art. The 3 5 anisotropy of resolution and sharpness has been discussed above.
Another effect of the anisotropy associated with prior art systems is the obvious uneven illumination of the image field. That is, one side ~ 7 ~

of the field of view appears bright while the opposite side appears dark. The introduction in the present invention of multiple beams makes it possible to produce an evenly illuminated field of view.

An evenly spaced four beam system (not shown) in which one pair of adjacent beams provides the illumination for one eyepiece and the other pair of adjacent beams provides the illumination for the other eyepiece, provides the advantage of overall high resolution inuring to a system of two beams at right angles, with 3-D viewing. It follows that a six beam system of two pairs of three beams would give even greater resolution for 3-D viewing.

The utilization in the present invention of a plurality of light beams following different paths to the condenser makes it possible to individually manipulate those beams for a variety of possible results in addition to enhanced resolution. For exammple, referring to Fig. 2, real time 3-D is achieved by interposing complimentary polarizing filters 36 and 37 in beam paths 28 and 32, respectively together with providing similar eyepiece polarizing filters 38 and 39 in binocular eyepiece 41 having a pair of viewing lenses 42 and 43. The filters 36 and 37 are denoted by - positive and negative symbols to indicate that they could be complementary in a variety of different ways known in the art. They may be plane polarizers oriented with their polarizing axes mutually at right angles. Alternatively, they may be circular polarizers, one of the pair producing left-hand polarization, the other producing right-hand polarization. Yet in another alternative, the filters may be complementary color filters ( such as red and green) of either the absorption or dichroic type. The eye piece filters 38 and 39 interact with filters 36 and 37 to selectively limit the light from only one of the light sources 26 and 29 so that the irnage produced by the light along beam path 33 does not exit the viewing lens 43.
and the image produced by the light along beam path 34 does not exit the viewing lens 42.
The overlap of the filtered bea.ms which is possible by adjustment of beam path shift means 23 and 24 creates real 3-D
o images and by being able to independently control the direction of the light paths of the bearns. it becomes possible to control the parallax angles for left and right images, and thereby control the degree of depth perception in the final image.
The present invention goes far beyond what can be achieved with a single beam. real time. 3-D system in which the degree of parallax is fixed.and there is very little disparity in parallax between the left and right images especially at the center of the image field.

wo 92/18X94 PC~/US92/02260 -18- 2~4408 In contrast. with the present invention the left and right images are independently controlled and the degree of parallax between them can be easily adjusted to match the type of objective being employed and the type of specimen being viewed. In addition, 5 there is another and possibly even more important advantage with the present invention. which is the ability to achieve a greater depth of field without loss of resolution. This is a critical prerequisite for producing a sharp 3-D image.
A microscope utili7ing the illumination system of the present 10 invention can use any of the many light beam manipulation devices known in microscopy, such as polari~ing filters, aperture stops.
collimator, etc. In multiple bearn systems of the present invention these devices can be used to provide beams having different characteristics or those having the same characteristics.
Since resolution is enhanced by oblique illumination primarily along the axis (in both directions) of the illl~min~ting light beam.
while being diminished along the axis 90 degrees thereto. a first order approximation of high resolution over the entire specimen plane is achieved using two beams. Adding more beams will further 20 enhance the distribution of high resolution over the specimen plane. However, little is gained by using more than 5 or six oblique light beams, radially spaced about the optical axis. As can be seen from the previous discussion about the anisotropy of resolution associated with a single oblique beam (figures lE and lF). the fall-25 off in resolution is negligible within 15 degrees or so either side ofthe axis of each illuminating beam (it is proportional to the cosine of that angle).
By way of example for a 3 beam system. referring to Figures 3, 3A and 3B, mirror surfaces 45. 46 and 47 are supported on beam 30 shift means 48, 49 and 50. respectively. Each mirror surface is disposed in one of the source beam paths 51, 52 and 53. of light beams 54. 56. and 57, respectively, emanating from light beam sources means 58. 59 and 61. The shift means 48. 49 and 50 as best seen with reference to Figures 3A and 3B. are movable along 35 paths 55 that are radial relative to the optical axis 13 of the wo 92/18894 PCr/uss2/0226u -19- 2~
condenser means 12 (see Figs. 2 and 3). For purposes of the present invention, the beam shift means are positioned at locations on their paths 55 that place the beam reflecting mirror surfaces 45, 46 and 47 radially out~vard from the axis 13. As fully described 5 above. varying the location of a mirror (45 for example) along its radial path 55 varies the angle of the beam exit path 66 (see Fig. 3) from condenser lens means 12.
Referring to ~igure 3C. "bright field" illllmination is available in the present system by locating one of the mirror surfaces (47 for 10 example) over the optical axis 13 and in a position in which it creates a beam path that travels along the condenser lens means axis 13. The other mirror surfaces can be deployed to provide oblique lighting at the same time or disabled (mirrors moved out of range of the condenser means or associated light beam source 15 means turned off) for standard "bright field" illl~min~tion.
Positioning of the shift means 48, 49 and 50 can also result in "dark field" illumination. When the radial location of the mirror surfaces create bearn exit paths from the condenser means that are angled to fall outside of the objective aperture. "dark field"
20 illumination is made possible.
Referring to Fig. 3, each mirror surface 45, 46 and 47 is also angularly tiltable relative to its associated source me~nS be~n so as to vary the angle of reflectance of its mirror surface. Thus. by tilting a mirror surface the angle of the beam path from the shift 25 means to the condenser means 12 is varied and in turn the location of the exit beam path from the condenser means is varied.
The source means bearns 54. 56 and 57 follow source beam paths 51, 52 and 53 to the light path shift means 48, 49 and 50 that are generally normal to the axis 13 of condenser meansl2 and 30 evenly angularly spaced about the axis 13 of the condenserl2 and the axis 16 of the objective lens means 14 which axes are shown as being coincident (see Fig. 2). The mirrors 45,46 and 47 are positionable radially and angularly to establish the direction of the beam paths 62, 63 and 64 to the condenser lens means 12, and 35 thereby control the location and direction of the exit paths 66, 67 wo 92/18894 Pcr/lJs92/o2~6o -20- 2 Q ~
and 68 from the condenser means to the objective means..
The practicalities of size and space between the shift means and the condenser lens means 12 makes it very difficult to gather all of the light from the individual beams 54, ~6 and 57 and direct 5 it onto condenser lens 12 at precisely the location and angle necessary to achieve the desired exit paths from condenser lens 12. A large field lens 71 (such as a 50 mm f/ 1.2 camera lens) acts as a pre-condenser lens means permitting the gathering of all of the light from the incident 10 beams and the accurate direction of those light beams onto the condenser lens 12. The raising or lowering of the field lens 71 relative to the condenser lens 12 has the effect of sizing the beam on the specimen plane 20 to accommodate low power as well as high power systems.
Furthermore, a field lens aperture (iris stop) 72 can be used to control depth of field and contrast, provided the condenser means 12 is slightly under focused. Prior art systems reduce the condenser lens aperture to increase depth of field but in doing so reduce resolution due to a concomitant reduction in the numerical 20 aperture of the light beam exiting the condenser. However, in the multiple beam embodiment of the present invention, the condenser aperture 69 rem~in~ fully open while the field lens aperture 72 can be reduced to increase depth of field without a concomitant loss in resolution. This is because the aperture of 25 each illuminating beam is reduced while the overall aperture of illumination that exits the condenser lens is not significantly reduced. The multiple beams illuminate the full condenser aperture and no loss of resolution is experienced.
The interposition of the field lens 71 and iris stop 72 does -30 not interfere with the operation of the present invention since adjustment of the mirror surfaces 45. 46, and 47 continues to control the direction and location of the exit paths of the beams from the condenser lens 12.
Likewise, the interposition in the source beam paths 54. 56 35 and 57 of such devices as lamp condensers 73. zoom lens 74 (to wo 92/1~894 Pcr/~S92/02260 20'8 !~
adjust beam size), and polarizing filters 76 does not interfere with the operation of the present invention and in fact highlights one of its major advantages. The use of such light manipulating devices on the light beams either separately (between the light source and the 5 shift means) or together, such as by the field lens 71, the field lens aperture (iris) 72 or a polarizing filter 77 ( between the shift means and an eye piece 78), does not reduce the system's resolution.
Where light sources 58, 59 and 61 are independent (as opposed to a single source split by optical means) they can be l O varied in intensity to add yet a further investigatory variation.
~ rom the forgoing it is apparent that in order to achieve enhanced resolution the present invention does not limit the use of well known optical devices for light manipulation nor does it result in operation at low light levels relative to the light provided by the 15 light source means. Thus, the illumination system of the present invention enhances resolution and at the same time makes it possible to create illumination conditions that can satisfy a wide variety of investigation needs.
A multi- beam system of the present invention enjoys 20 enhanced resolution both from an increase in the oblique orientation of the illuminating beams relative to the objective lens means optical axis (increase in orders of wavelets recombined) as well as from an increase in the overall aperture of illllmin~tion of the condenser lens due to the additive effect of the multiple light 25 beams that exit the condenser from around its periphery.
When polarizing filters 76 in the source beam paths 5~, 56 and 57 from the light source means to the beam shift means are complementary, rotation of polarizing filter 77 in the combined beam between the objective lens meansl4 and the eyepiece lens 78 30 permits rotation of the shadow effect of the oblique lighting on the specimen by ef~ectively attenuating the illumination from one or two of the beams while looking at the effects of the other.
The present invention is independent of any particular mechanical or electrical system for positioning and directing the 35 illuminating beams. This includes systems that may be adjustable wo 92/1~894 Pcr/~S92/02~60 -22- ~ ~ ~
or pre-adjusted and fixed and may utilize mirrors. prisms fiber optics or other known or unknown devices. Such mechanical systems can take any number of forms known to those skilled in the art. By way of example. such a mechanical arrangement for 5 positioning the mirrors of the shift means is described with reference to Figure 4.
A shift means 80 includes a mirror 81 affixed to a tilt arrn 82 which is rotatably connected by hinge 83 to an "L" shaped mount member 84 which is secured to a car 86 that runs in tracks 87. A
10 cable 88 attached to a tab 89 formed on the end of mount member 84 provides the means for positioning the car 86 on the trac~ 87 and thus the radial position of the mirror 81 relative to an optical axis. A pivot arm 91 is pivotally attached at one of its ends to the tilt arrn 82 and at its other end to a slide 92 that runs in a groove 15 93 in the mount member. A cable 94 affixed to a tab 96 formed in the end of slide 92 positions the slide in its groove and in doing so adjusts the tilt of the tilt arm 82 and the angle of the mirror 81.
The use of micrometers (not shown) attached to the ends of the cables 94 and 98 to operate the cables makes it possible to achieve 20 the degree of precision necessary for the invention.
A number of shift means 80 in the same system can be mechanically inter-connected (by means well within the skill of the art) so that their positions will be inter-dependent. That is. the movement of one shift means to a new radial location or the tilting 25 of a mirror to a different angular position will cause corresponding movement in the other shift means. This arrangement sets a fixed relationship between the mirrors and makes it possible to easily assure that all of the beams are substantially identical in their paths through the system other than their circumferential position 30 relative to the objective axis 16.
Where it is desired to be able to vary one bearn path without disturbing the others. then the positioning of the shift means is most advantageously mechanically independent. In the preferred embodiment of the invention the mirror members are selectively 35 mechanically inter-connected for unified movement and - 23 - D 2 ~ ~ 4 ~ ~ ~

mechanically unconnected ~or independent movement. Such a system is capable of satisfying the needs of a wide variety of microscope uses.
The same mechanical apparatus described above for positioning mirrors can be used to position fiber optic guides directing light onto the condenser as shown in Figure 6.
Once again, the present invention is independent of any particular mechanical system for interconnecting the beam shift means which mechanical systems can take any number of forms known to those skilled in the art.
The method of the present invention for increasing resolution. sharpness and depth of field in a transmitted light microscope having a condenser lens means with an optical axis.
and an objective lens means with an optical axis. which is apparent from the forgoing, constitutes the steps of directing a plurality of independent light beams onto the condenser lens means along paths that are not coincident with the condenser lens means optical axis: and fixing the location and direction of the paths of the light beams to the condenser lens means so that the light beams that exit the condenser lens means are directed along paths that include the objective lens means and are oblique relative the optical axis of the objective lens means. Further, the directions of the paths of the light beams onto the condenser lens means are selected to produce exit paths from the condenser lens that are at the optimal angle relative to the optical axis of the objective lens means that includes the objective lens means.
When the number of beams is two and they are directed along paths that are in opposition to one another (essentially 180 degrees apart) they provide the best illumination for real-time 3-D viewing.
When they are at right angles (90 degrees to one an other) they 4 ~ ~
-- 24 _ provide overall resolution using ju~t two beams. When the number of beams is three or more they are preferably radially positioned and spaced about the optical axis of the condenser lens means for the best overall resolution at the specimen plane.
The invention having been fully described, it is not limited to the details herein set forth. but is of the full scope of the appended claims.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An illumination system for a transmitted light microscope including a condenser lens means having an optical axis and objective lens means having an optical axis, comprising in combination:
light beam source means located off the optical axis of the condenser lens means for providing a light beam along a source beam path that passes through the condenser lens means; and beam path shift means operable to shift the source beam path to be non-parallel to the optical axis of the condenser lens means.

2. The invention of claim 1 wherein said light beam source means provides a plurality of independent light beams along source beam paths that pass through the condenser lens means, and said beam path shift means shifts a plurality of source beam paths.

3. An illumination system for a transmitted light microscope including a condenser lens means having an optical axis and objective lens means for having an optical axis, comprising in combination:
light beam source means providing a light beam along a source beam path that is non-coincident with the optical axis of the condenser lens means; and beam path shift means disposed off the optical axis of the condenser lens means in said source beam path, said beam path shift means operable to shift the path of said source means light beam and redirect it along an incident path that passes through the condenser lens means, and an exit path from the condenser lens means that passes through the objective lens means, wherein the path between the condenser lens means and the objective lens means is oblique to the optical axis of the objective lens means.

4. The invention of claim 3 wherein said shift means is operable to variably alter the direction of the path of the light beam to the condenser lens means over a range that produces exit paths from the condenser lens means that passes through the objective lens means.

5. The invention of claim 3 wherein said light beam shift lens means is operable to variably alter both the location and the angle of the exit beam path from the condenser lens means.

6. The invention of claim 5 wherein said light beam shift means is operable to independently vary the location and the angle of the exit beam path.

7. The invention of claim 6 wherein said light beam shift means comprises a mirror angularly moveable relative to said source means light beam, and laterally movable relative to the optical axis of the condenser lens means.

8. The invention of claim 6 wherein the condenser lens means includes a pre-condenser lens.

9. The invention of claim 8 wherein the pre-condenser lens means further includes a field stop iris.

10. An illumination system for a transmitted light microscope including a condenser lens means having an optical axis, and an objective lens means having an optical axis, comprising in combination:
light beam source means for providing a plurality of independent light beams along different source beam paths which are non-coincident with the optical axis of the condenser lens means; and a plurality of light beam shift means each of which is operable to shift the path of a source means light beam and redirect it along an incident path that passes through the condenser lens means, and an exit path from the condenser lens means that passes through the objective lens means, wherein the exit path is oblique to the optical axis of the objective lens means.

11. The invention of claim 10 wherein said light beam shift means are operable to variably alter the direction of the path of their associated light beam to the condenser lens means over a range that produces exit paths from the condenser means that passes through the objective lens means.

12. The invention of claim 11 wherein said light beam shift means are operable to variably alter both the location and angle of the exit beam paths from the condenser.

13. The invention of claim 11 wherein said light beam shift means are independently operable.

14. The invention of claim 11 wherein said light beam shift means are operably interconnected whereby the operation of one to vary the path of its associated light beam causes a variation in the path of the light beam associated with each of the other light beam shift means.

15. The invention of claim 10 wherein each of said light beam shift means is fixed to create a single exit path from said condenser lens means.

16. The invention of claim 10 wherein the condenser lens means includes a pre-condenser lens optically disposed in the incident paths of beams from said shift means to the condenser lens means.

17. The invention of claim 16 wherein said pre-condenser lens is physically movable relative to said condenser lens means.

18. The invention of claim 16 wherein said pre-condenser lens means further includes an associated field stop iris.

19. The invention of claim 10 wherein the number of light beams and the number of said light beam shift means is two.

20. The invention of claim 19 wherein the illumination system further includes means for supporting a specimen at a specimen plane between the condenser means and the objective means, and wherein said shift lens means are operable to establish light beam paths which at the specimen plane are at essentially right angles to each other.

21. The invention of claim 19 wherein the illumination system further includes means for supporting a specimen at a specimen plane between the condenser lens means and the objective lens means, and wherein said light beams are essentially 180 degrees apart in opposing relationship to one another at the location of the specimen plane.

22. The invention of claim 19 further comprising complementary filters disposed between said source means and condenser lens means.

23. The invention of claim 22 wherein said filters are plane polarizers.

24. The invention of claim 22 wherein said filters are circular polarizers.

25. The invention of claim 22 wherein said filters are complementary colors.

26. The invention of claim 10 wherein the number of independent light beams and the number of said light beam shift means is three.

27. The invention of claim 26 wherein the illumination system further includes means for supporting a specimen at a specimen plane between the condenser lens means and the objective lens means and wherein said exit beam paths are evenly angularly spaced from one another at the specimen plane.

28. The invention of claim 26 wherein the system further includes an eyepiece lens means disposed to receive light from the objective lens means and wherein the invention further comprises polarizing filters optically disposed between said source means and said condenser lens means and a polarizing filter optically disposed between the objective lens means and the eyepiece lens means.

29. The invention of claim 26 further comprising a zoom lens optically disposed between said source means and said shift means.

30. The invention of claim 26 wherein said beam path shift means comprises three mirrors disposed about the optical axis of the condenser lens means and in the source beam paths, said mirrors movable along paths that are radial relative to the optical axis of the condenser lens means and angular relative to the source beam paths.

31. The invention of claim 30 wherein the movement of one of said mirrors is independent of the others.

32. The invention of claim 30 wherein the movement of one of said mirrors causes movement in the others.

33. An illumination system for a transmitted light microscope including a condenser lens means having an optical axis, and an objective lens means having an optical axis, comprising in combination:

light beam source means for providing a plurality of independent light beams along different source beam paths to the condenser lens means;
a plurality of mirror surfaces optically disposed in source beam paths wherein each said mirror surface is movable radially relative to the optical axis of the condenser means and angularly relative to its associated source beam, wherein said mirror surfaces are movable to positions along radial paths in which they do not intersect the condenser means optical axis whereby said mirror surfaces are operable to direct source beams onto the condenser lens means at locations thereon which are determined by the radial and angular positions of said mirror surfaces.

34. The invention of claim 33 wherein at least one of said mirror surfaces is movable radially to a position in which said mirror surface intersects the optical axis of the condenser means.

35. The invention of claim 33 in which the movement of said mirror surfaces is interdependent on each other.

36. The invention of claim 33 in which the movement of said mirror surfaces is independent of each other.

37. The invention of claim 33 in which the movement of said mirror surfaces is selectably independent of each other and interdependent on each other.

38. The invention of claim 33 wherein the range of movement of each mirror surface produces a path that passes through the objective lens means.

39. The invention of claim 33 wherein the range of movement of each mirror surface produces paths that pass through and pass by the objective lens means.

40. The invention of claim 33 wherein the condenser lens means includes a pre-condenser leans means.

41. The invention of claim 40 wherein the pre-condenser lens means further includes a field stop iris.

42. A method of increasing resolution in a transmitted light microscope having a condenser lens means having an optical axis, and an objective lens means having an optical axis, comprising the steps of:
directing a plurality of independent light beams onto the condenser lens means along paths that are not coincident with the condenser lens means optical axis;
adjusting the direction of the paths of the light beams to the condenser lens means until the light beams that exit the condenser lens means are directed along paths that pass through the objective lens means and are oblique relative the optical axis of the objective lens means.

43. The method of claim 42 wherein the directions of the paths of the light beams onto the condenser lens means are adjusted until the light beams that exit the condenser lens means are directed along paths that are at the maximum angle relative to the optical axis of the objective lens means and also pass through the objective lens means.

44. The method of claim 42 wherein the number of beams is two.

45. The method of claim 44 wherein the beams are directed along paths that are essentially 90 degrees apart.

46. The method of claim 44 wherein the beams are directed along paths that are essentially 180 degrees apart.

47. The method of claim 42 wherein the number of beams is more than two and they are positioned circumferentially about the optical axis of the condenser lens means.

48. A method for real-time 3-D imaging with increased resolution in a transmitted light microscope having a condenser lens means having an optical axis, an objective lens means having an optical axis, a source means of source light beams and a binocular eyepiece means, comprising the steps of:
directing two independent light beams from the source means into the condenser lens means along paths that are not coincident with the condenser lens means optical axis and which produce exit beam paths from the condenser lens means which are oblique to the optical axis of the objective lens means and which pass through the objective lens means, disposing complementary filters between the source means and the condenser lens means; and disposing another set of complimentary filters on the binocular eyepiece means.

49. The method of claim 48 further comprising the step of adjusting the location and angle of the source beam path between the source means and the condenser means to control parallax.

50. The method of claim 48 wherein the condenser lens means is further described as including a pre-condenser lens and iris diaphragm having an iris aperture, further comprising the step of adjusting the iris aperture to adjust depth of field.

51. The method of claim 10 wherein said light beam shift means include mirror surfaces.

52. The method of claim 10 wherein said light beam shift means include fiber optics.

53. The method of claim 10 wherein the number of independent light beams is four.

54. The method of claim 10 wherein the number of independent light beams is six.

55. The method of claim 22 wherein the system further includes an eyepiece lens means disposed to receive light from the objective lens means and wherein the invention further comprises complementary filters optically disposed between the objective lens means and the eyepiece lens means.

56. The method of claim 26 wherein the different source beam paths include fiber optic means and said light beam shift means includes fiber optic position means.

57. In an illumination system for a transmitted light microscope having a condenser lens means with an optical axis, and an objective lens means with an optical axis, the combination comprising:

light means providing a plurality of independent light beams having paths which are oblique with respect to the optical axis of the condenser lens means, and which pass through both the condenser lens means and the objective lens means, wherein the paths of the beams between the condenser lens means and the objective lens means are oblique to the optical axis of the objective lens means.

58. The method of claim 57 wherein the number of beams is 3.

59. The method of claim 57 wherein the number of beams is 4.

60. The method of claim 57 wherein the number of beams is 6.

61. In an illumination system for a transmitted light microscope having a condenser lens means with an optical axis, and an objective lens means with an optical axis, the combination comprising:
light means providing a plurality of independent light beams each having an optical axis which lies along a path which is non-coincident with the optical axis of the condenser lens means, which independent light beams overlap at the condenser lens means and which independent light beams pass through both the condenser lens means and the objective lens means, wherein the paths of the optical axis of the independent light beams between the condenser lens means and the objective lens means are oblique to the optical axis of the objective lens means.

62. The invention of claim 61 wherein the number of independent light beams is 3.

63. The invention of claim 61 wherein the number of independent light beams is 4.

64. The invention of claim 61 wherein the number of independent light beams is 6.